A VALIDATED REVERSED PHASE HPLC ASSAY FOR THE DETERMINATION OF MEFENAMIC ACID IN HUMAN PLASMA

Nada H. Binhashim and Muhammad M. Hammami*

ABSTRACT

A simple and precise reversed-phase high performance liquid chromatographic (HPLC) assay for the measurement of mefenamic acid level in human plasma, using diclofenac as an internal standard (IS), was developed and validated. Plasma samples containing mefenamic acid were spiked with the IS then extracted with acetonitrile and reconstituted in mobile phase. The compounds of interest were efficiently separated on Atlantis dC18 column at room temperature and detected with photodiode array detector set at 278 nm. The mobile phase consisted of 0.025 M dibasic potassium phosphate (pH = 6.0, adjusted with phosphoric acid) and acetonitrile (65:35, v:v) and was delivered at a flow rate of 1.5 ml/min. The relationship between mefenamic acid concentration in plasma and peak area ratio of mefenamic acid/ IS was linear (R2  0.9987) in the range of 0.05 – 10 μg/ml, the intra- and inter- day coefficient of variations (CV) were ≤ 5.3% and ≤ 7.2%, respectively, and the intra- and inter- day bias was ≤ 6% and ≤ 8%, respectively. Mean extraction recovery of mefenamic acid and the IS from plasma samples was 99% and 92%, respectively. The method was used to assess the stability of mefenamic acid in human plasma under various conditions encountered in the clinical laboratory. Mefenamic acid was stable for 8 weeks at -20°C (≥ 95%), 24 hours at room temperature (≥ 96%) and after 3 freeze-thaw cycles (≥ 96%) in unprocessed plasma samples, and for 48 hours at -20°C (≥ 90%) and 24 hours at room temperature (≥ 92%) in processed samples.

Keywords: Mefenamic acid, Diclofenac, Human plasma, HPLC.